The Nodaviridae have bipartite RNA genomes, that is to say their genomes consist of two separate single-stranded RNA molecules, designated RNA1 and RNA2. These are both packaged within the same virion. RNA1 encodes an RNA replicase, and RNA2 encodes the virion capsid protein. In flock house virus (FHV), RNA1 is 3.1 kb long and RNA2 is 1.4 kb.
The replicase product of FHV RNA1 is specific for the viral genome and this enables FHV to replicate autonomously [Ball et al. (1992) J. Virol. 66:2326–34: Gallagher et al. (1983) J. Virol. 46:481–89]. In a natural situation, the replicase is highly template specific and replicates only viral RNA1 and RNA2, but self-replication also occurs in the absence of RNA2. Furthermore, even though FHV is an insect virus, it can self-replicate in many different cell types, including plants, vertebrates and yeasts.
Manipulation of RNA2 and the FHV capsid protein has been widely reported. The insertion of HIV epitopes into surface loops has been reported [e.g. Scodeller et al. (1995) Vaccine 13:1233–39; Buratti et al. (1996) J. Immunol. Methods 197:7–18; Schiappacassi et al. (1997) J. Virol. Methods 63:121–27]. More generally, the virion has been used as an epitope display system [Lorenzi & Burrone (1999) Immunotechnol. 4:267–72; see also WO96/05293].
In contrast, manipulation of FHV RNA1 and the replicase has not been pursued. In fact, the self-replication function of RNA1 has been shown to be very sensitive to manipulation of RNA1 and its ORF [Ball (1995) J. Virol 69:720–727].
During replication of RNA1, a small sub-genomic RNA called RNA3 is also transcribed from the 3′ end of RNA1, RNA3 encodes for two small proteins of unknown function: B1 (in the same open reading frame and with the same translational stop codon as the replicase) and B2 (in the +1 open reading frame with respect to the replicase) [Ball (1992) J. Virol 66:2335–45; Johnson & Ball (1999) J. Virol. 73:7933–79421. Transcription of RNA3 seems to be controlled by an internal promoter which becomes active when a double stranded RNA+/RNA− intermediate is formed. It is an object of invention to permit modification and manipulation of the RNA1 molecule to exploit its ability to self-replicate, and to provide such modified RNA1 molecules.